Project Summary/Abstract Mammarenaviruses establish chronic infections in their natural rodent reservoirs across the world, and mammarenavirus zoonoses can pose important public health problems in their endemic regions. Mammarenaviruses can subvert the innate immune responses in infected individuals, thus compromising the development of an effective antiviral adaptive immune response, which facilitates unrestricted virus multiplication and associated pathological manifestations and disease. The mammarenavirus lymphocytic choriomeningitis virus (LCMV) provides us with a highly tractable experimental system to elucidate virus-host immune system interactions contributing to these events. We have documented that LCMV nucleoprotein (NP), as well as NPs from other mammarenaviruses, inhibits production of interferon ? (IFN?), a key player in the host innate immune defense against viral infections. The type I IFN (IFN-I) counteracting activity of mammarenavirus NP correlated with the NP's ability to inhibit activation of IRF3 and NF-kB. Arenavirus NP's anti-IFN-I activity was mapped to the C-terminal region of NP that contains a functional 3'-5' exonuclease (ExoN) domain and an overlapping IKK?-interacting domain. The experiments in this application are designed to elucidate the following issues: 1) whether arenavirus NP's ability to inhibit induction of IFN? production requires its binding to IKK? or its ExoN activity, or both; and, 2) the mechanisms by which mutations affecting NP-IKK? interaction or NP's ExoN activity affect virus fitness and whether this result in the loss of LCMV's ability to persist in its natural host, the mouse. To this end, we propose to complete the following specific aims: 1. Determine whether NP-IKK? interaction is required for NP-mediated inhibition of IFN? induction in LCMV- infected cells: We will identify residues within arenavirus NP that are required for NP-IKK? interaction, and functionally characterize NP mutants impaired in their ability to associate with NP-IKK?, including their ExoN activity and ability to counteract induction of IFN?. 2. Evaluate the contribution of the ExoN activity of arenavirus NP to inhibition of IFN? induction and virus fitness in the absence of a functional IFN-I system: We will conduct mutation-function studies to determine whether the ExoN activity of NP is strictly required for its anti-IFN? activity, and to promote normal LCMV multiplication in the absence of a host cell functional IFN-I system. These studies will examine the role of the ExoN activity of NP viral transcription and LCMV replication fidelity. 3. Roles of NP's anti-IFN-I and ExoN activities in virulence and the establishment of the natural carrier state of LCMV in mice: We will use reverse genetics to generate rLCMVs carrying the different types of NP mutations. These rLCMVs will be first confirmed to exhibit their predicted phenotypes in cultured cells, and then will be examined for their in vivo phenotypic features in the context of LCMV infection of its natural host, the mouse.